Inappropriate activation of certain cellular proteins is associated with the development of various human disease, including cancer, cardiovascular, immunological and neurological diseases. R. J. Mayer, Protein Degradation: The Ubiquitin-Proteasome System and Disease, Volume 4, Wiley-VCH, 2007. For example, in many cancers, overexpression of one or more oncogenes plays a critical role in the initiation and progression of tumors. C. M. Croce, Oncogenes and Cancer, N. Engl. J. Med. 2008; 358:502-511. Therefore, an effective method to down-regulate the level of a target protein which causes or contributes to a disease would have significant diagnostic and therapeutic impacts. Additionally, different tools have been harnessed to reduce target protein levels in order to assess their biological functions.
RNA interference (RNAi), gene knockout, ribozymes and anti-sense oligonucleotides are frequently used in different eukaryotic organisms to eliminate or reduce the level of a cellular protein. However, in some cases, these techniques do not sufficiently ablate protein expression. Oftentimes, because existing gene products are removed only at the rate of their natural turnover, the immediate depletion of a target protein cannot be achieved. Another factor to consider is the stability of the target protein, which dictates the rate and, therefore, the efficiency of its depletion. Even if nascent mRNAs have been totally destructed, the residual long-lived target proteins may distort or obscure the assessment of the protein ablation phenotype.
Ubiquitin-dependent proteolysis is a major catabolic pathway utilized by eukaryotic cells for the degradation of cellular proteins. Protein ubiquitination is catalyzed by concerted actions of three classes of enzymes: the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin protein ligases (reviewed in Hochstrasser (1996) Annu Rev. Genet 30: 405 39). While E1 and E2 are primarily involved in the activation and transfer of ubiquitin, the substrate specificity of the ubiquitin pathway is conferred by the E3 ubiquitin protein ligases. Recently, we demonstrated that, through a technique we designated “protein knockout” (PKO), engineered ubiquitin ligases can be utilized to accelerate proteolysis of specific intracellular proteins. Zhou et al. Harnessing the ubiquitination machinery to target the degradation of specific cellular proteins. Mol. Cell. 2000; 6: 751-6. Zhang et al. Exploring the functional complexity of cellular proteins by protein knockout. Proc Natl Acad Sci USA 2003; 100: 14127-32. Zhang et al. Ectopic targeting of substrates to the ubiquitin pathway. Methods Enzymol 2005; 399: 823-33. Zhou P. Targeted protein degradation. Curr Opin Chem Biol 2005; 9: 51-5.
By combining RNAi and protein knockout techniques, the Applicant has further developed a method that reduces the amount of a target protein rapidly and effectively. RNAi operates at the level of protein biosynthesis via degradation of the specific mRNA or inhibition of its translation, while ubiquitin ligase-mediated protein knockout functions at the post-translational level to accelerate the degradation of the desired protein. This combination approach is generally applicable to any target protein, including proteins that cannot be sufficiently eliminated by RNAi, and proteins with a relatively long half-life.